Teses / dissertações sobre o tema "ATF6α"

Mutations in the COL10A1 gene cause metaphyseal chondrodysplasia type Schmid (MCDS) by triggering ER stress and unfolded protein response (UPR). MCDS is characterised by a mild short-limb dwarfism accompanied by expansion of the cartilage growth plate hypertrophic zone (HZ) and altered differentiation of hypertrophic chondrocytes (HCs). ATF6 is one of the UPR mediators, which exists in two isoforms, ATF6α and ATF6β. Activation and up-regulation of ATF6α was a prominent biochemical sign of ER stress in a mouse model of MCDS, COL10a1 p.N617K. Although ATF6β is induced and activated in response to ER stress in a similar fashion to ATF6α, the role and significance of ATF6β in the pathology of many ER stress-associated diseases including MCDS is unknown. Here we utilized a combination of in vitro and in vivo approaches to define the precise role of each isoform of ATF6 in MCDS.To investigate the functions of ATF6α and ATF6β in vitro, we developed a MCDS cell model system (expressing either the wild type collagen X or one of the following MCDS-causing mutant forms of the protein: p.N617K, G618V, Y598D, and NC1del10) in which the expression of either ATF6α or ATF6β was efficiently silenced using siRNAs. ATF6α knockdown in HeLa cells expressing different MCDS-causing mutations suppressed the increased expression of UPR-associated genes such as BiP leading to an elevated ER stress, based on increased XBP1 splicing and/or ATF4 protein. In contrast, ATF6β knockdown did not significantly affect the mutant collagen X-induced increased expression of UPR-associated genes. Furthermore, the ER stress levels were significantly reduced in the ATF6β knockdown MCDS mutant cells based on the lower levels of XBP1 splicing and/or ATF4 protein detected. We then crossed the ATF6α/β knockout mice models with COL10a1 p.N617K mouse model of MCDS to investigate the function of ATF6α and ATF6β in vivo. Ablation of ATF6α in MCDS mice further- reduced the endochondral bone growth rate, further expanded the growth plate hypertrophic zone, and disrupted differentiation of HCs. Therefore, ATF6α appeared to play a chondroprotective role in MCDS as its deficiency caused an increase in the severity of the disease. Of particular note, the level of ER stress was further increased in the absence of ATF6α in MCDS, based on enhanced activities of PERK and IRE1 signalling pathways in compensation for the ATF6α loss. Paradoxically, ablation of ATF6β in MCDS mice reduced the intracellular retention of collagen X protein, and alleviated the ER stress as judged by the attenuated activities of PERK and IRE1 signalling pathways. The reduced ER stress resulting from deficiency for ATF6β in MCDS mice restored the expression of collagen X mRNA towards normal and improved the differentiation of HCs, causing a mark decrease in the expansion of HZ. The results presented within this thesis greatly increased our understanding of the function of ATF6α and ATF6β and their interplay in the pathogenesis of MCDS. We demonstrated an indispensable beneficiary role for ATF6α but a detrimental role for its closely related isoform, ATF6β, in pathology of MCDS. We also showed that the role of ATF6β should not be ignored. These findings may be used to develop a potential therapeutic strategy for MCDS through targeting and enhancing ATF6α-dependent and/or attenuating/blocking of ATF6β-dependent signalling pathways.

Contexte : En France, la prévalence des modifications corporelles apparentes augmente, y compris chez les médecins généralistes. Il n’existe actuellement pas d’étude sur le vécu des médecins porteurs de modifications corporelles apparentes dans leur relation avec le patient.Méthode : Etude qualitative par IPA par entretiens semi-directifs entre janvier et juin 2024. Le recrutement a eu lieu au cours du CNGE de 2022, par « effet boule de neige » et via la diffusion d’annonces sur les réseaux sociaux. 6 médecins ont été interrogés sur la base d’un guide d’entretien révisé après chaque entretien.Résultats : Dans l’imaginaire collectif le look du médecin généraliste reste stéréotypé et est un moyen de se présenter à l’autre. Le médecin a tendance à s’auto-censurer dans son look pour se conformer à la norme sociale tout en ayant le désir d’être authentique. Les patients accueillent de façon bienveillante le plus souvent les modifications corporelles apparentes. Certains sont plus réticents mais les compétences professionnelles semblent primer sur la première impression. Les médecins s’amusent parfois des réactions des patients, d’autant plus s’ils assument pleinement leurs modifications corporelles. Pour d’autres, le bilan est plus mitigé, notamment lorsqu’un manque de légitimité se fait ressentir.Conclusion : En l’absence d’autre facteur de discrimination, les modifications corporelles apparentes du médecin généraliste sont plus ou moins acceptables selon leur visibilité et la sensibilité, la « norme » du patient. Il serait d’intérêt de réaliser d’autres études quant à l’impact des différents facteurs de discrimination du médecin sur la relation médecin-patient.Le vieillissement cutané, influencé par une combinaison de facteurs intrinsèques et extrinsèques, entraine des dommages capables d'altérer les fonctions cutanées. Parmi les facteurs extrinsèques, les rayonnements ultraviolets (UV) sont responsables du photo-vieillissement de la peau. Ces éléments conduisent notamment à une accumulation de cellules sénescentes capables de contribuer au développement de pathologies liées à l’âge, telles que les cancers cutanés. En effet, la sénescence s’accompagne de profonds changements morphologiques et moléculaires au sein de la cellule. Cela inclut notamment une modification de son sécrétome, qui s'enrichit en cytokines pro-inflammatoires, en facteurs de croissance et en enzymes remodelant la matrice extracellulaire, altérants les caractéristiques des tissus lors du vieillissement. Néanmoins, les mécanismes précis qui aboutissent au phénotype sénescent induit par les UVB restent largement inconnus. Dans ce contexte, l’objectif principal de ce travail a été d'identifier des mécanismes moléculaires sous-jacents à l’établissement de la sénescence induite par les UVB dans des fibroblastes de derme humains normaux (NHDFs), mécanismes qui pourraient contribuer au vieillissement cutané. In vitro, nous avons confirmé que des expositions répétées aux UVB induisent la sénescence prématurée des NHDFs et que cet état est associé à l’activation des trois branches de la voie UPR (Unfolded Protein Response) responsables du maintien de l’homéostasie du réticulum endoplasmique (RE), le premier compartiment de sécrétion. Ces observations ont été supportées par une analyse transcriptomique, révélant des éléments de régulation liés aux grandes voies de sénescence et aux fonctions du RE dans les NHDFs exposés aux UVB. Par la suite, nous avons montré que la branche ATF6α joue un rôle central dans la survenue des biomarqueurs du phénotype sénescent induit par les UVB. En effet, l’invalidation d’ATF6α protège non seulement des changements morphologiques induits par les UVB, mais réduit le pourcentage de cellules positives pour la SA-βgalactosidase (SA-βgal), prévient la persistance des dommages à l'ADN, et modifie l'expression de facteurs majeurs du phénotype sécrétoire associé à la sénescence (SASP) [...]<br>Skin ageing, influenced by a combination of intrinsic and extrinsic factors, can result in damage that has the potential to alter skin functions. Among extrinsic factors, ultraviolet (UV) radiation is responsible for skin photoageing. These factors notably contribute to the accumulation of senescent cells which in turn can contribute to the development of age-related pathologies, including skin cancers. Indeed, senescence is characterized by profound morphological and molecular changes within the cell. This includes a modification of its secretome, which becomes enriched in pro-inflammatory cytokines, growth factors, and matrix-remodelling enzymes, altering tissue characteristics during ageing. However, the exact mechanisms driving the senescent phenotype induced by UVB remain largely unknown. In this context, the main objective of this work was to identify the underlying molecular mechanisms responsible for the establishment of UVB-induced senescence in normal human dermal fibroblasts (NHDFs), mechanisms that may play a role in skin ageing. In vitro, we confirmed that repeated exposures to UVB induce premature senescence of NHDFs and that this state is associated with the activation of the three branches of the Unfolded Protein Response (UPR), which are responsible for maintaining endoplasmic reticulum (ER) homeostasis, the primary cellular secretion compartment. These observations were supported by transcriptomic analysis, revealing regulatory elements related to major senescence pathways and ER functions in UVB-exposed NHDFs. Subsequently, we demonstrated that the ATF6α branch plays a central role in the development of the UVB-induced senescent phenotype. Indeed, the silencing of ATF6α not only protects against morphological changes induced by UVB, but also reduces the percentage of senescence-associated β-galactosidase (SA-βgal) positive cells, prevents the persistence of DNA damage, and alters the expression of major factors associated with the senescence-associated secretory phenotype (SASP). The SASP, exerting a pro-tumoral action, led us to assess whether the conditioned medium (CM) from UVB-exposed fibroblasts invalidated for ATF6α could impact the migration and invasion potential of melanoma cells. However, we did not observe any ATF6α-dependent pro-migratory or pro-invasive effects. To highlight a potential role of ATF6α in another biological process, we further analyzed our transcriptomic and secretomic analyses and identified a possible effect of ATF6α on the paracrine control of the skin environment. To explore this, we focused on SASP factors (cytokines and metalloproteinases) regulated by ATF6α and whose impact on tissue environment was known. Subsequently, we treated a reconstructed human epidermis (RHE) model with CM from NHDFs exposed or not to UVB and invalidated or not for ATF6α.Surprisingly, we observed that the CM from UVB-exposed NHDFs increased the thickness of the RHE as well as the proliferation of basal keratinocytes, via an ATF6α-dependent mechanism. Finally, we identified IL-8 as a major paracrine factor involved in this process, as blocking IL-8 with neutralizing antibodies prevented excessive proliferation of keratinocytes. In conclusion, we report the role of ATF6α in UVB-induced senescence and its impact on the preservation of skin homeostasis under stress conditions, particularly through the regulation of the expression of SASP components. This suggests that ATF6α and its effectors could be promising targets for controlling the effects of skin ageing

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. Tumor progression is strictly related to ER stress, while cancer cells are prone to tolerate unfolded protein accumulation and to take advantages from ER stress-related pro-survival pathways. Mutation of Tp53 gene is a frequent event in human tumor and a significant factor in cancer development and progression. We report that cancer cells bearing mutant p53 respond to ER stress insult by dampening ER-stress associated pro-apoptotic factor and by sustaining survival. Mechanistically, we find that mutp53 is inhibiting JNK and CHOP and is promoting ATF6 transcriptional activity. These observations reveals a protective role of mutant p53 in the response to chronic ER stress, offering an additional perspective to cancer treatment. Indeed, we observed a cooperative effect in using mutp53 and ATF6 inhibitors in killing cancer cells.

La mucoviscidose est la maladie génétique létale à transmission autosomale récessive la plus fréquemment retrouvée dans la population européenne. Elle est due à des mutations altérant le gène CFTR, dont la plus fréquente est la mutation induisant la délétion d’une phénylalanine en position 508 de la séquence polypeptidique de cette protéine (p.Phe508del-CFTR). Ces mutations altèrent la viscosité du mucus présent à la surface apicale des cellules épithéliales des systèmes respiratoire, digestif et génital. Cela entraîne une baisse de la clairance mucociliaire, rendant difficile le renouvellement de ce mucus qui est la première barrière protectrice vis-à-vis du développement de microorganismes potentiellement pathogènes. En conséquence, des réponses inflammatoire et infectieuse se mettent en place. En ajoutant l’accumulation de protéines mal repliées dans la lumière du RE, le mécanisme de défense adaptatif UPR est déclenché. ATF6 fait partie de ses trois voies régulatrices. Il a été montré que ATF6 inhibait l’expression du CFTR. Le but de ce projet de thèse est d’évaluer les effets de l’inhibition de la S1P, une protéine centrale pour l’activation de ATF6, sur le p.Phe508del-CFTR par des moyens pharmacologiques. Les résultats montrent que les efflux d’ions Cl- lié à l’activité du canal p.Phe508del-CFTR était augmenté grâce à une augmentation de l’expression globale, ainsi que du transport de ce canal jusqu’à la membrane plasmique. Nous donnons également quelques pistes pouvant expliquer ces effets bénéfiques, notamment en rapport avec le déclenchement de l’UPS qui est une voie permettant le transport de protéines mutées jusqu’à la membrane plasmique<br>Cystic fibrosis is the most common lethal autosomal recessive genetic disease in the European population. It is caused by mutations in the CFTR gene, the most common of which is the deletion of a phenylalanine at position 508 of the protein's polypeptide sequence (p.Phe508del- CFTR). These mutations alter the viscosity of the mucus present on the apical surface of epithelial cells in the respiratory, digestive and genital systems. This leads to a reduction in mucociliary clearance, making it difficult to renew the mucus that forms the first protective barrier against the development of potentially pathogenic micro- organisms. As a result, inflammatory and infectious responses are triggered. By adding the accumulation of misfolded proteins in the lumen of the ER, the UPR adaptive defence mechanism is triggered. ATF6 is one of its three regulatory pathways. ATF6 has been shown to inhibit CFTR expression. The aim of this thesis project is to evaluate the effects of inhibiting S1P, a protein central for the activation of ATF6, on p.Phe508del-CFTR by pharmacological means. The results show that Cl- ion efflux linked to the activity of the p.Phe508del-CFTR channel is increased through an increase in the overall expression and transport of this channel to the plasma membrane. We also give some possible explanations for these beneficial effects, in particular in relation to the triggering of the UPS, a pathway that allows mutated proteins to be transported to the plasma membrane

La mucoviscidose est une maladie causée par des mutations du gène cftr entraînant des défauts importants de la protéine CFTR. La mutation la plus fréquente (F508del) est caractérisée par un repliement incorrect conduisant à la rétention de la protéine dans le RE.L’accumulation de CFTR-F508del dans le RE, l’inflammation et les infections vont déclencher un stress du RE dans les cellules épithéliales ainsi que l’UPR. Cette dernière est une réponse adaptative déclenchée par le stress du RE et permet de rétablir l’homéostasie de ce compartiment. L’UPR est constituée de trois voies majeures dont l’une d’entre elles est activée dans les cellules exprimant un CFTR-F508del. Il s’agit de la voie ATF6 qui est de plus responsable de la répression transcriptionnelle du CFTR, ce qui en fait une cible thérapeutique potentielle. Nous avons montré que son inhibition conduit à l’amélioration de la fonction duCFTR-F508del et à l’augmentation de sa présence à la membrane des cellules.Nous nous sommes également intéressés au Mg2+ et au TRPM7, le régulateur principal de la [Mg2+]i dans les cellules. Nous avons émis l’hypothèse que TRPM7 était en partie responsable de l’activation d’ATF6 dans les cellules exprimant un CFTR-F508del. Le but de cette seconde partie du projet était donc tout d’abord d’étudier la relation existante entre le Mg2+, TRPM7 et le CFTR. Nous avons montré qu’il existait des différences de [Mg2+]i selon le type de mutation du CFTR exprimé par les cellules. Ces différences sont en partie dues à un défaut d’activation de TRPM7, lui-même probablement lié à un défaut du CFTR. En augmentant l’activité de TRPM7 par du Naltriben, nous avons pu montrer un effet potentialisant sur leCFTR-G551D<br>Cystic fibrosis is caused by mutations in the cftr gene resulting in several defaults on the CFTR protein. The most frequent mutation is F508del which is characterized by an incorrect folding causing its retention within the ER. CFTR-F508del protein accumulation in the ER, inflammation and infections will trigger the ER stress in epithelial cells, as well as UPR. UPR constitutes an adaptive response of the ER in order to restore ER’s homeostasis. UPR consists in three major pathways. Among them, one is activated in cells expressing CFTR-F508del protein. The ATF6 pathway of UPR is responsible of the transcriptional repression of CFTR, which makes of it a potential therapeutic target. We showed that the inhibition of ATF6 leads to the improvement of CFTR-508del function, as well as its increased presence in the cellular membrane. We were also interested in Mg2+ and TRPM7, the main regulator of [Mg2+]i. We suspected that TRPM7 is, at least in part, responsible for the activation of ATF6 in cells expressing the mutant CFTR-F508del. Thus, the second part of my work was focused on the study of the relationship between Mg2+, TRPM7 and CFTR. We showed the existence of [Mg2+]I differences according to CFTR mutant expressed in cells. These differences are the result of an altered TRPM7 activation, probably in link with the mutated CFTR’s malfunction. We proved that increasing TRPM7 activity by Naltriben treatment potentiates CFTR-G551D

La présente thèse explore le monde de la biologie du stress du RE (réticulum endoplasmique). Une vue globale du RE et du stress du RE est d'abord fournie en commençant par les mécanismes de base impliqués pour aller vers de possibles applications cliniques. L'accent est ensuite mis sur le rôle crucial de l'UPR dans la cancérogénèse, qui est activée en réponse au stress du RE dans la micro-environnement de la tumeur. Après avoir passé en revue ces aspects, nous mettons en évidence des éléments manquants dans notre compréhension de la façon dont les signaux UPR sont affinés et conduisent soit à la restauration de l'homéostasie du RE et des cellules soit à la mort cellulaire. Parmi les branches de l'UPR, les signaux ATF6 et IRE1 deviennent notre sujet d'investigation en raison de leur convergence dans la régulation du facteur XBP1 favorisant la survie. D'une part, nous découvrons les mécanismes provenant du lumen du RE qui régulent l'activation de l'ATF6 en réponse au stress du RE et affectant la signalisation adaptative cellulaire de l'ATF6 en aval. D'autre part, nous observons l'existence d'un réseau autorégulateur de l'activité RNase de l’IRE1 consistant en un système tyrosine kinase-phosphatase ciblant la RtcB et impactant l'épissage de l'ARNm de XBP1. Ainsi, grâce à nos études, nous avons découvert un circuit de signalisation intégré capable d’ajuster avec précision les sorties cellulaires de l’activation conjointe ATF6 et IRE1 en réponse au stress du RE<br>The present thesis explores the world of ER (endoplasmic reticulum) stress biology. A global view of ER and ER stress is first provided with a transition from the basic mechanisms involved to possible clinical applications. The focus is then placed to the crucial role of the UPR in carcinogenesis that is activated in response to ER stress in the micro-environment of the tumor. After reviewing these aspects, we point to missing parts in our comprehension of how UPR signals are fine-tuned and lead to either restoration of ER and cell homeostasis or cell death. Among the UPR branches, ATF6 and IRE1 signaling become our focus of investigation because of their convergence in the regulation of the pro-survival factor XBP1s. On the one hand, we unravel mechanisms originating from the ER lumen that regulate the ATF6 activation in response to ER stress and affect its downstream cell adaptive signaling. On the other hand, we witness the existence of an auto-regulatory network of IRE1 RNase activity consisted of a tyrosine kinase-phosphatase system that targets RtcB and impacts on XBP1 mRNA splicing. Hence, through our studies we uncover an integrated signaling circuit that can fine-tune the cellular outputs of the joint ATF6 and IRE1 activation in response to ER stress

The establishment of non-resolving inflammation underlies the pathogenesis of chronic inflammatory diseases in humans. Super low dose (SLD) endotoxin has been associated with exacerbating inflammation and the pathogenesis of chronic inflammatory diseases. However, the underlying molecular mechanisms are not well studied. In this study, I tested the hypothesis that SLD endotoxin may potentiate non-resolving innate immune cell inflammation through disrupting cellular endoplasmic reticulum (ER) homeostasis. We chose to study the dynamics of ER homeostasis in macrophages stimulated with SLD endotoxin. In naïve cells, ER stressor such as tunicamycin (TM) not only will induce cellular stress and inflammation through JNK and NFkβ activation, but also will cause subsequent compensatory homeostasis through inducing homeostatic molecules such as XBP1 and GRP78/BiP. We observed that cells challenged with SLD endotoxin have significantly reduced expression of homeostatic molecules XBP1 and BiP. Mechanistically, we observed that SLD-LPS increases phosphorylated HCK expression in TM treated cells. Phosphorylated HCK activation resulted in the phosphorylation of Golgi protein GRASP, leading to unstacking of Golgi cisterna and overall dysfunction of the Golgi apparatus. Dysfunctional Golgi apparatus and its effect on protein transport and secretion, may account for decreased levels of Site 2 Protease, reduced generation of ATF6 and its transcriptional target BiP. Taken together, our study reveal that super low dose endotoxin exacerbates low grade inflammation through increasing phosphorylation of HCK, inducing Golgi dysfunction, and decreasing BiP /homeostatic protein expression in innate immune cells.<br>Ph. D.

Endoplasmic Reticulum Stress (ERS) is one hallmark of cancer cells: tumor hypoxia, glucose reduction and genome instability all promote accumulation of misfolded proteins in the endoplasmic reticulum. ER stress triggers the Unfolded Protein Response (UPR), a conserved pathway initiated by three ER-resident receptors, IRE1α, PERK, and ATF6, that activate specific and overlapping transcriptional programs aimed to overcome the stress or induce cell death. Accumulating evidence suggest a role for UPR in cancer progression, therefore uncovering functional interactions of this pathway with the oncogenic circuits that drive various tumors may be relevant for therapy. The tumor suppressor p53 is one of the most frequently mutated genes in cancer and missense mutant p53 proteins (mutp53) can acquire powerful oncogenic properties. We recently reported that mutant p53 can modulate the UPR, specifically sustaining activation of the ATF6 branch. This molecular axis may contribute to cancer aggressiveness and resistance to therapy. However, the mechanisms by which mutant p53 can modulate the UPR in cancer cells remained unexplored. In this Thesis, I describe one of the possible mechanisms exploited by mutp53 to sustain ATF6. Using breast, prostate and mammary cancer cell lines, I found that mutant p53 enhances ERS-induced activation of the stress kinase p38MAPK. I also found that inhibition of p38MAPK reduces ERS-induced proteolytic cleavage of ATF6 and its transcriptional activity. These data suggest that p38MAPK may have a pro-survival role in the context of ER stress. Indeed, pharmacologic inhibition of p38MAPK increased the sensitivity to Thapsigargin in cancer cells with mutant p53. Regarding the possible action of p38, I measured the turnover of the active ATF6 fragment, and found that inhibition of p38MAPK induced a perceptible reduction in ATF6f stability. Therefore, one mechanism by which mutp53 can reshape the UPR is by increasing the stability of the active ATF6f protein via enhanced activation of p38MAPK.

This research was originally published in The Journal of Biological Chemistry. Satoshi Horimoto, Satoshi Ninagawa, Tetsuya Okada, Hibiki Koba, Takehiro Sugimoto, Yukiko Kamiya, Koichi Kato, Shunichi Takeda, and Kazutoshi Mori. The Unfolded Protein Response Transducer ATF6 Represents a Novel Transmembrane-type Endoplasmic Reticulum-associated Degradation Substrate Requiring Both Mannose Trimming and SEL1L Protein. The Journal of Biological Chemistry. 2013. 288:31517-31527. © the American Society for Biochemistry and Molecular Biology<br>Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(理学)<br>甲第19543号<br>理博第4203号<br>新制||理||1603(附属図書館)<br>32579<br>京都大学大学院理学研究科生物科学専攻<br>(主査)教授 森 和俊, 教授 阿形 清和, 准教授 細川 暢子<br>学位規則第4条第1項該当

Aging is associated with a loss of proliferation of the insulin-secreting beta cell, a possible contributing factor to the greatly increased rate of type-2 diabetes in the elderly. A landmark study from our lab previously illustrated that mild endoplasmic reticulum (ER) stress drives beta cell proliferation specifically through ATF6α, one arm of the tripartite Unfolded Protein Response (UPR). It is unknown if old beta cells differ from young beta cells in UPR signaling or proliferative response to ER stress or ATF6α activation. To investigate, young and old mouse islets were cultured ex vivo in high glucose, and beta cell proliferation was quantified by BrdU incorporation after treatment with low dose thapsigargin or activation of overexpressed ATF6α. In addition, levels of UPR signaling were compared by semi-quantitative Xbp1 splicing assay. Interestingly, although old beta cells displayed reduced proliferation in glucose compared to young beta cells, their proliferative response to low-dose thapsigargin and ATF6α activation were nearly identical, and no difference was found in Xbp1 splicing under high glucose or high ER stress conditions. These results suggest that the aged mouse beta cell does not have impaired UPR-responsive proliferation or aberrant UPR signaling when cultured ex vivo

Background: NUPR1 was described as a transcriptional factor involved in the regulation of various cellular stress-response genes, playing a crucial role in the condition of the endoplasmic-reticulum (ER) stress, thus emerging as a common molecular factor of different pathologies, obesity, hepatic steatosis, and cancer. In the present work we aim to explore how NUPR1 interacts with some pivotal genes that are the major modulators of the ER stress and metabolic cell functions. In particular we investigated the biochemical and molecular effects arising from the loss of NUPR1 in ER stress physiological conditions. Methods: We used prolonged high fat diet (HFD) feeding to induce ER stress physiological in Nupr1+/+ and Nupr1-/- male mice compared with their respectively normal chow diet (ND) controls. We fed mice with a HFD (60% fat, 20% protein, and 20% carbohydrate) for 10 weeks to promote chronic ER stress condition (Old-HFD group, n=5). An additional group of mice (n=5) was maintained on HFD (60% fat, 20% protein, and 20% carbohydrate) for a longer duration (15 weeks) to distinguish between age-dependent and age-independent effects. Liver were collected for histological and molecular assessments. Western blots and RT-qPCR were performed to assess the expression levels of the major ER-stress response UPR-associated proteins and metabolic genes. Results: We showed the downregulation of the majority of UPR-associated proteins: BIP (p<0.0001 for protein and mRNA), ATF4 (p<0.0001 for mRNA), XBP1 (p<0.0001 for protein and mRNA), CHOP (p<0.0001 for protein and mRNA), GADD34 (p=0.0296 for mRNA) in in-vivo NUPR1-/- compared to NUPR1+/+ 10 weeks HFD mice. Western blot for the major UPR associated proteins in NURP1-/- mice at 15 weeks HFD showed similar expression trends reported at the time-point of 10 weeks. ERDj4 mRNA resulted down-regulated in NUPR1-/- compared to NUPR1+/+ 15 weeks HFD mice (p=0.0032). Among the multiple metabolic genes, we reported a down-regulation of the majority mRNA associated to lipogenesis (SREBP, ACLY, ChREBP) and lipoprotein (APOB, PPAR-alfa, MTTP) in NUPR1-/- compared to NUPR1+/ + HFD mice 15 weeks. Both LCAD and MCAD fatty acid metabolisms mRNA were also downregulated, as consequence of PPAR-alfa deficit. Similarly betaoxidation mRNA ACOX1 and CPT1-alfa, as well as MTC4 and PGK1 were downregulated in NUPR1-/- compared to NUPR1+/ + HFD mice 15 weeks. Conclusion: The results of this work confirm that NUPR1 act downstream of the PERK branch playing a crucial role of NUPR1 in the activation of UPR response in physio-pathological ER stress condition and suggest a potential contribution of NUPR1-mediated ER stress response to the development of liver steatosis.<br>Background: NUPR1 was described as a transcriptional factor involved in the regulation of various cellular stress-response genes, playing a crucial role in the condition of the endoplasmic-reticulum (ER) stress, thus emerging as a common molecular factor of different pathologies, obesity, hepatic steatosis, and cancer. In the present work we aim to explore how NUPR1 interacts with some pivotal genes that are the major modulators of the ER stress and metabolic cell functions. In particular we investigated the biochemical and molecular effects arising from the loss of NUPR1 in ER stress physiological conditions. Methods: We used prolonged high fat diet (HFD) feeding to induce ER stress physiological in Nupr1+/+ and Nupr1-/- male mice compared with their respectively normal chow diet (ND) controls. We fed mice with a HFD (60% fat, 20% protein, and 20% carbohydrate) for 10 weeks to promote chronic ER stress condition (Old-HFD group, n=5). An additional group of mice (n=5) was maintained on HFD (60% fat, 20% protein, and 20% carbohydrate) for a longer duration (15 weeks) to distinguish between age-dependent and age-independent effects. Liver were collected for histological and molecular assessments. Western blots and RT-qPCR were performed to assess the expression levels of the major ER-stress response UPR-associated proteins and metabolic genes. Results: We showed the downregulation of the majority of UPR-associated proteins: BIP (p<0.0001 for protein and mRNA), ATF4 (p<0.0001 for mRNA), XBP1 (p<0.0001 for protein and mRNA), CHOP (p<0.0001 for protein and mRNA), GADD34 (p=0.0296 for mRNA) in in-vivo NUPR1-/- compared to NUPR1+/+ 10 weeks HFD mice. Western blot for the major UPR associated proteins in NURP1-/- mice at 15 weeks HFD showed similar expression trends reported at the time-point of 10 weeks. ERDj4 mRNA resulted down-regulated in NUPR1-/- compared to NUPR1+/+ 15 weeks HFD mice (p=0.0032). Among the multiple metabolic genes, we reported a down-regulation of the majority mRNA associated to lipogenesis (SREBP, ACLY, ChREBP) and lipoprotein (APOB, PPAR-alfa, MTTP) in NUPR1-/- compared to NUPR1+/ + HFD mice 15 weeks. Both LCAD and MCAD fatty acid metabolisms mRNA were also downregulated, as consequence of PPAR-alfa deficit. Similarly betaoxidation mRNA ACOX1 and CPT1-alfa, as well as MTC4 and PGK1 were downregulated in NUPR1-/- compared to NUPR1+/ + HFD mice 15 weeks. Conclusion: The results of this work confirm that NUPR1 act downstream of the PERK branch playing a crucial role of NUPR1 in the activation of UPR response in physio-pathological ER stress condition and suggest a potential contribution of NUPR1-mediated ER stress response to the development of liver steatosis.

We have identified a novel Cyclin, called Cyclin O, which is able to bind and activate Cdk2 in response to intrinsic apoptotic stimuli. We have focused on the study of Cyclin O&#945; and Cyclin O&#946;, alternatively spliced products of the gene. Upon treatment with different stress stimuli, transfected Cyclin O&#945; accumulates in dense aggregations in the cytoplasm compatible with being Stress Granules (SGs). Furthermore, we have seen that Cyclin O&#946; and a point mutant of the N-terminal part of the protein constitutively localize to the SGs. Although both alpha and beta isoforms are proapoptotic, only Cyclin O&#945; can bind and activate Cdk2. On the other hand, we have demonstrated that Cyclin O is upregulated by Endoplasmic Reticulum (ER) stress and is necessary for ER stress-induced apoptosis. Cyclin O activates specifically the PERK pathway and interacts with the PERK inhibitor protein p58IPK. Moreover, Cyclin O participates in the activation of other eIF2&#945; kinases. We have also observed that a pool of Cyclin O is located in active mitochondria, suggesting a function of the protein linked to oxidative metabolism.<br/><br/>Hemos identificado una nueva Ciclina, llamada Ciclina O, que es capaz de unirse y activar Cdk2 en respuesta a estímulos apoptóticos intrínsecos. Nos hemos centrado en el estudio de la Ciclina O&#945; y la Ciclina O&#946;, productos de splicing alternativo del gen. En respuesta a diferentes tipos de estrés, la Ciclina O&#945; se acumula en agregaciones citoplásmicas densas que podrían corresponder a Gránulos de Estrés (SGs). Además, hemos visto que la Ciclina O&#946; y un mutante puntual de la parte N-terminal de la proteína se localizan constitutivamente en los SGs. Aunque las dos isoformas alfa y beta son proapoptóticas, solo la Ciclina O&#945; es capaz de unirse y activar Cdk2. Por otro lado, hemos demostrado que los niveles de Ciclina O se incrementan en respuesta al estrés de Retículo Endoplásmico (RE) y que esta proteína es necesaria para la inducción de apoptosis dependiente de estrés de RE. La Ciclina O activa específicamente la vía de PERK e interacciona con la proteína inhibidora de PERK p58IPK. Además, la Ciclina O participa en la activación de otras quinasas de eIF2&#945;. La Ciclina O se localiza en mitocondrias activas, lo que sugiere una función de la proteína ligada al metabolismo oxidativo.

Endoplasmic reticulum (ER) stress has been implicated as a causative factor in the development of pancreatic beta-cell dysfunction and death resulting in type 2 diabetes. This thesis examined the role of ATF6beta in the ER stress response of beta-cells. Using an ATF6beta-specific antibody, expression of full-length ATF6beta was detected in various insulinoma cell lines and rodent islets and the induction of the active form (ATF6beta-p60) under ER stress conditions. Knock-down of ATF6beta in INS-1 832/13 cells did not affect mRNA induction of known ER stress response genes in response to tunicamycin-induced ER stress, however it increased the susceptibility of beta-cells to apoptosis. Conversely, overexpression of ATF6beta-p60 reduced the apoptotic phenotype. Microarray results suggest ATF6beta functions to induce expression of adaptive genes also regulated by ATF6alpha, but also several specific targets genes. These findings have increased our understanding of the role of ATF6beta in the ER stress response of beta-cells.

Aging remains to this day one of the unresolved biology areas of upmost importance and many age-related diseases are on the rise worldwide. One of aging major hallmarks, proteostasis, has several associated pathways across different segments which have not yet been fully detailed in various cell lines and are needed to better understand the underlying aging problem. Here, using a neuronal-like cell line such as SH-SY5Y, several ER stress biomarkers and proteostasis associated targets are evaluated under ER stress-induced environment through tunicamycin (TUN) or thapsigargin (TG) presence. The inclusion of neuroprotective agents such as TUDCA and homegrown compounds (HA compounds) were also included to better evaluate successful chemical reversion of ER stress and protein aggregation through target proteins. BAG3, ATF4, Calreticulin and pERK1/2 were some of the proteins included in this report as biomarkers for ER stress induction using protein or gene expression level analysis. ER stress was effectively induced with thapsigargin or tunicamycin across all target proteins. ATF4, calreticulin and pERK1/2 protein and/or gene expression values decreased after neuroprotective agents’ treatment. However, no ER stress reversion was achieved for GRP78 and BAG3. XBP1s achieved positive results only for tunicamycin-treated conditions. Overall, ER stress induction was partially or totally reverted with success by TUDCA and HA compounds in SH-SY5Y.<br>O envelhecimento permanece até hoje uma das áreas biológicas por resolver de maior importância. Muitas doenças associadas ao envelhecimento estão a aumentar de forma global. Uma característica principal associada ao envelhecimento é a proteostase, cujos diferentes componentes ainda não foram totalmente descritos em diferentes linhas celulares. Aqui, usando um modelo celular neuronal como as células SH-SY5Y, diversos biomarcadores de stress do retículo endoplasmático e de agregação proteica foram avaliados em ambientes de stress do RE induzidos por tunicamicina ou tapsigargina, bem como a sua reversão. A inclusão de agentes protetores (TUDCA) e reversores químicos da agregação proteica (compostos HA) foram incluídos para melhor avaliar essa mesma reversão do stress do RE. BAG3, ATF4, calreticulina e pERK1/2 foram algumas das proteínas incluídas nesta dissertação e a avaliação do stress do RE foi alcançada pela análise dos seus níveis de expressão proteicos e/ou génicos. A indução do stress do RE foi alcançada eficazmente tanto para a tapsigargina como para a tunicamicina, em todas as proteínas-alvo, nesta linha celular. ATF4, calreticulina e pERK1/2 foram diminuídas pela ação dos agentes protetores e, consequentemente, diminuiu o stress do RE. No entanto, para a GRP78 e BAG3, não se obtiveram resultados de reversão do stress do RE. XBP1s apenas alcançou resultados significativos de reversão no caso das condições tratadas com tunicamicina. Em suma, o stress do RE induzido por TG ou TUN foram revertidos parcialmente ou na sua totalidade com sucesso pelos agentes protetores nesta linha celular.<br>Mestrado em Biomedicina Molecular

碩士<br>國立清華大學<br>生物科技研究所<br>95<br>Geldanamycin (GA), a benzoquinone ansamycin, is an inhibitor of heat shock protein 90 (HSP90)/ glucose-regulated protein 94 (GRP94) and has been implicated as a potent anti-cancer drug. In our previous study, we found that GA with sublethal dose provoked the ER stress in 9L rat brain tumor (RBT) cells and induced glucose-regulated proteins under unfold protein response (UPR) at transcriptional level. The promoter of grp genes contain multiple copies of the ER stress response element (ERSE), with a consensus of CCAAT(N9)CCACG, which is critical and necessary for transcription induction. Herein, we showed that GRP94, an ER resident chaperone, was induced under GA treatment in 9L rat brain tumor (RBT) cells and the mRNA level of grp94 was peaked at 8 h for about 9 fold. We analyzed the promoter sequence of grp94 according to rat genome resource and designed reporter vectors containing progressive-deleted promoters of grp94. In reporter gene assays, ERSE4 and CRE-BP1/c-Jun played the major roles in GA-induced grp94 expression. Moreover, with mutagenesis clones we further confirmed this result although other ERSEs still have involved in. By inhibitors screening, pretreatment of KT5720, BIM I, or Gö6983 partially decreased GA-induced GRP94 expression, suggesting the involvement of PKA and PKC. Pretreatment of AEBSF blocked proteolysis of ATF6 abolished GA-induced GRP94 expression. Taken together, we found that under GA induced ER stress in 9L cells, activation of GRP94 were mainly through ERSEs by the transcription factor ATF6.

Pancreatic beta-cells are responsible for secreting insulin into the circulation to maintain whole body glucose homeostasis. While pancreatic beta-cells have a large capacity to secrete insulin, their function progressively deteriorates during the pathogenesis of type 2 diabetes as a result of both genetic predisposition and environmental factors. Obesity is the largest risk factor for developing type 2 diabetes and is associated with various conditions that can impair normal beta-cell function, including excess free fatty acids, inflammation and insulin resistance. Accumulating evidence in the literature suggests that endoplasmic reticulum (ER) stress contributes to the molecular mechanism of pancreatic beta-cell failure during the progression of type 2 diabetes. In this thesis, I have examined the role of the ER stress sensor ATF6-alpha and also the ER-resident chaperone GRP78 in pancreatic beta-cell homeostasis and function. Work presented in Chapter 2 examined the function of naturally occurring ATF6-alpha protein variants associated with type 2 diabetes. I also examined the role of endogenous ATF6-alpha in pancreatic beta-cells, which is described in Chapter 3. Results from these analyses suggest that the ATF6-alpha gene is not a type 2 diabetes susceptibility gene; however, ATF6-alpha protein expression is important to beta-cell function and survival. Finally, ER stress markers have been detected in pancreatic beta-cells and insulin sensitive tissues (such as adipose and liver), which promote beta-cell dysfunction and insulin resistance, respectively. In Chapter 4, I examined the contribution of ER stress in beta-cell dysfunction specifically by generating transgenic mice over-expressing GRP78. The mice were subsequently challenged by high fat diet to determine their susceptibility to developing symptoms of type 2 diabetes. Indeed increased chaperone capacity in pancreatic beta-cells protected against obesity-induced glucose intolerance and insulin resistance. Overall, these data support the hypothesis that ER stress contributes to beta-cell dysfunction in type 2 diabetes progression.

Le diabète de type 2 (DT2) est caractérisé par une résistance des tissus périphériques à l’action de l’insuline et par une insuffisance de la sécrétion d’insuline par les cellules β du pancréas. Différents facteurs tels que le stress du réticulum endoplasmique (RE) et l’immunité innée affectent la fonction de la cellule β-pancréatique. Toutefois, leur implication dans la régulation de la transcription du gène de l’insuline demeure imprécise. Le but de cette thèse était d’identifier et de caractériser le rôle du stress du RE et de l’immunité innée dans la régulation de la transcription du gène de l’insuline. Les cellules β-pancréatiques ont un RE très développé, conséquence de leur fonction spécialisée de biosynthèse et de sécrétion d’insuline. Cette particularité les rend très susceptible au stress du RE qui se met en place lors de l’accumulation de protéines mal repliées dans la lumière du RE. Nous avons montré qu’ATF6 (de l’anglais, activating transcription factor 6), un facteur de transcription impliqué dans la réponse au stress du RE, lie directement la boîte A5 de la région promotrice du gène de l’insuline dans les îlots de Langerhans isolés de rat. Nous avons également montré que la surexpression de la forme active d’ATF6α, mais pas ATF6β, réprime l’activité du promoteur de l’insuline. Toutefois, la mutation ou l’absence de la boîte A5 ne préviennent pas l’inhibition de l’activité promotrice du gène de l’insuline par ATF6. Ces résultats montrent qu’ATF6 se lie directement au promoteur du gène de l’insuline, mais que cette liaison ne semble pas contribuer à son activité répressive. Il a été suggéré que le microbiome intestinal joue un rôle dans le développement du DT2. Les patients diabétiques présentent des concentrations plasmatiques élevées de lipopolysaccharides (LPS) qui affectent la fonction de la cellule β-pancréatique. Nous avons montré que l’exposition aux LPS entraîne une réduction de la transcription du gène de l’insuline dans les îlots de Langerhans de rats, de souris et humains. Cette répression du gène de l’insuline par les LPS est associée à une diminution des niveaux d’ARNms de gènes clés de la cellule β-pancréatique, soit PDX-1 (de l’anglais, pancreatic duodenal homeobox 1) et MafA (de l’anglais, mammalian homologue of avian MafA/L-Maf). En utilisant un modèle de souris déficientes pour le récepteur TLR4 (de l’anglais, Toll-like receptor), nous avons montré que les effets délétères des LPS sur l’expression du gène de l’insuline sollicitent le récepteur de TLR4. Nous avons également montré que l’inhibition de la voie NF-kB entraîne une restauration des niveaux messagers de l’insuline en réponse à une exposition aux LPS dans les îlots de Langerhans de rat. Ainsi, nos résultats montrent que les LPS inhibent le gène de l’insuline dans les cellules β-pancréatiques via un mécanisme moléculaire dépendant du récepteur TLR4 et de la voie NF-kB. Ces observations suggèrent ainsi un rôle pour le microbiome intestinal dans la fonction de la cellule β du pancréas. Collectivement, ces résultats nous permettent de mieux comprendre les mécanismes moléculaires impliqués dans la répression du gène de l'insuline en réponse aux divers changements survenant de façon précoce dans l’évolution du diabète de type 2 et d'identifier des cibles thérapeutiques potentielles qui permettraient de prévenir ou ralentir la détérioration de l'homéostasie glycémique au cours de cette maladie, qui affecte plus de deux millions de Canadiens.<br>Type 2 diabetes is characterized by insulin resistance and impaired insulin secretion from the pancreatic β-cell. Endoplasmic reticulum (ER) stress and innate immunity have both been reported to alter pancreatic β-cell function. However, it is not clear whether these factors can affect the transcription of the insulin gene. The aim of this thesis was to assess the role of ER stress and innate immunity in the regulation of the insulin gene. Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. In a first study, using several approaches we showed that ATF6 (activating transcription factor 6), a protein implicated in the ER stress response, directly binds to the A5/Core of the insulin gene promoter in isolated rat islets. We also showed that overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibits the activity of an insulin promoter-reporter construct. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity. In recent years, the gut microbiota was proposed has an environmental factor increasing the risk of type 2 diabetes. Subjects with diabetes have higher circulating levels of lipopolysaccharides (LPS) than non-diabetic patients. Recent observations suggest that the signalling cascade activated by LPS binding to Toll-Like Receptor 4 (TLR4) exerts deleterious effects on pancreatic β-cell function; however, the molecular mechanisms of these effects are incompletely understood. We showed that exposure of isolated human, rat and mouse islets of Langerhans to LPS dose-dependently reduced insulin gene expression. This was associated in mouse and rat islets with decreased mRNA expression of two key transcription factors of the insulin gene, PDX-1 (pancreatic duodenal homeobox 1) and MafA (mammalian homologue of avian MafA/L-Maf). LPS repression of insulin, PDX-1 and MafA expression was not observed in islets from TLR4-deficient mice and was completely prevented in rat islets by inhibition of the NF-kB signalling pathway. These results demonstrate that LPS inhibits β-cell gene expression in a TLR4-dependent manner and via NF-kB signaling in pancreatic islets, suggesting a novel mechanism by which the gut microbiota might affect pancreatic β-cell function. Our findings provide a better understanding of the molecular mechanisms underlying insulin gene repression in type 2 diabetes, and suggest potential therapeutic targets that might prevent or delay the decline of β-cell function in the course of type 2 diabetes, which affects more than two million Canadians.